The art of organic chemistry on solid supports is generally known. A useful review article on this topic may be found in “Organic Chemistry on Solid Supports” by Früchtel et al., Angew. Chem. Int. Ed. Engl., 1996, 35, pgs. 17–42, the contents of which are hereby incorporated by reference.
As discussed in Früchtel et al., the art has developed automated solid-phase synthesis of polypeptides, oligonucleotides and oligosaccharaides. Of particular interest here is solid-phase synthesis of oligonucleotides. The following are useful review articles/textbooks on this topic:    Beaucage et al., Tetrahedron, 1992, 48, 2223;    Davis et al., Innovation and Perspectives in Solid Phase Synthesis (Ed.; R. Epton), Intercept, Andover, 1992, pg. 63;    Montserra et al., Tetrahedron, 1994, 50, 2617; and    S. L. Beaucage et al., Tetrahedron, 1993, 49, 6123–6194;the contents of each of which are hereby incorporated by reference.
In the solid-phase synthesis of oligonucleotides, it is known to synthesize the oligonucleotide on an inorganic solid support bearing a succinyl linker arm—see, for example, any of the following references:    Caruthers et al., Genetic Engineering, Plenum Press, New York (1982), Vol. 4, pgs. 1–17;    Letsinger et al., Genetic Engineering, Plenum Press, New York (1985), Vol. 5, pg. 191;    Froehler et al., Nucleic Acids Research, 14:5399–5407 (1986); and    Matteucci et al., Journal of American Chemical Society, 103:3185–3186 (1981);the contents of each of which are hereby incorporated by reference.
Typically, the succinyl linker arm has the following general formula:
Thus, the succinyl group links the growing oligonucleotide from its terminal 3′ hydroxyl group by an ester bond to a primary amine on the support, which may be, for example, conventional controlled pore glass (CPG) or silica, by an amide bond. Once the desired oligonucleotide has been synthesized, it is freed or cleaved from the succinyl linker arm hydrolyzing the ester carbonyl group. The hydrolysis agent is usually concentrated ammonium hydroxide. Typically, this reaction can take from 1–4 hours to complete. With improvements to current solid-phase oligonucleotide synthesizers, this cleavage step can represent 50% or more of the total time require to synthesize the desired oligonucleotide.
Another type of linker arm is disclosed in U.S. Pat. No. 5,112,962 [Letsinger et al. (Letsinger)], the contents of which are hereby incorporated by reference. Letsinger teaches a linker arm for solid support synthesis of oligonucleotides and oligonucleotide derivatives have the following formula:
Thus, Letsinger teaches an oxalyl linker arm which purportedly release the synthesized oligonucleotide or oligonucleotide derivate in a period of 1–30 minutes in a manner that leaves the oligonucleotide fully protected. The oxalyl linker arm purportedly can be rapidly cleaved by 5% ammonium hydroxide in methanol, ammonium hydroxide, wet tertiary amine, triethylamine/alcohol, triethylamine/methanol, triethylamine/ethanol, aqueous trimethylamine and other bases. Unfortunately, the oxalyl linker arm of Letsinger suffers from its purported advantage. Specifically, the present inventors have discovered that the oxalyl linker arm of Letsinger is susceptible to significant spontaneous hydrolysis (e.g. spontaneous hydrolysis of ˜10–40% per month) which renders it difficult to use in commercial operations. The oxalyl arm is also difficult to prepare because it requires using oxalyl chloride, which is highly reactive, toxic and therefore dangerous.
Regardless of the specific nature of the linker arm, it is generally accepted in the art that the linker arm is not reusable after production and cleavage of the desired oligonucleotide. Thus, conventional linker arms may be regarded as non-recyclable. This is illustrated in FIG. 1 which illustrates the conventional use of a succinyl linker arm for the production of an oligonucleotide. Thus, as illustrated, after cleavage of the desired oligonucleotide, the support is irreversibly linked to the linker compound (i.e., the succinyl moiety) and cannot be reused.
The art is in need of a linker arm for solid support oligonucleotide synthesis, which linker arm is recyclable. More specifically, the art is in need of a linker arm capable of repeated oligonucleotide synthesis/cleavage.
In published International patent application WO 97/23496 [Pon et al.], the contents of which are hereby incorporated by reference, there is reported the first recyclable linker arm. This linker arm is based on a derivatized solid support having the following formula:
wherein: R8 is selected from the group consisting of a substituted or unsubstituted C1–C20 alkyl group, a substituted or unsubstituted C5–C30 aryl group and a substituted or unsubstituted C5–C40 alkylaryl group; X3 and X4 are the same or different and are selected from the group consisting of —O—, —S—, —S(O)2— and —N(R12)—; R12 is selected from the group consisting of a substituted or unsubstituted C1–C20 alkyl group a substituted or unsubstituted C5–C30 aryl group and a substituted or unsubstituted C5–C40 alkylaryl group; and Y is selected from the group consisting of:                —CH2—CH2—; —CH2—;        —CH2—O—CH2—; —CH2—CH2—CH2—;        —CH═CH—; —CH═C(CH3)—;        —C(CH3)═C(CH3)—; —CH2—C(═CH2)—; and        —CH2—S—CH2—.        
While a linker arm based on the solid support described by Pon et al. is a significant advance in the art, there is still room for improvement. Specifically, the solid support described by Pon et al. has the following disadvantages.
First, prior to attachment of the linker moiety, the solid support must be derivatized by a process comprising the step of reacting together the compounds of Formulae I, II and III:
wherein R8, X3, X4 and Y are as defined above. Practically, this involves two steps—i.e., reaction of the compound of Formula III with one of the compounds of Formulae I and II and subsequent reaction with the other of compounds of Formulae I and II. Thus, the disadvantage is additional labour required to effect a two-step derivatization of the solid support.
Second, each step of the derivatization described in the previous paragraph has the potential of incompletely derivatizing each HX4-moiety on the support thereby increasing the likelihood of a heterogeneous surface. Practically, it becomes necessary to block or cap underivatized HX4-moieties so that the linker moiety does interact with them. Thus, the disadvantage is additional labour and cost required to effect derivatization of the solid support.
Third, a linker arm based on the derivatized support described by Pon et al. is not as resistant to partial cleavage during regeneration as a derivatized support having a more fully saturated moiety.
In light of these disadvantages, it would be desirable to have an improved recyclable solid state support material useful in the oligonucleotide synthesis. It would be especially desirable if the the linker moiety could be attached to the support material with little or no derivatization required of the latter.